Combination therapy with semaphorin-4d blockade (sema4d) and dc1 therapy

ABSTRACT

Disclosed are compositions and methods comprising the administration of pulsed dendritic cells and an immunoregulator molecule inhibitor for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This is a non-provisional of pending U.S. provisional application Ser.No. 62/865,027, filed Jun. 21, 2019, the entirety of which applicationis incorporated by reference herein.

BACKGROUND

The aggressive features of various cancer types are mainly driven byoncodrivers such as HER2, HERS, EGFR, c-MET that are critically involvedin cell growth, proliferation, survival and differentiation.Overexpression of these oncodrivers has associated with poor prognosisand a key player in tumor cell resistance to targeted therapies. Ductalcarcinoma in situ (DCIS) is an early form of BC Stage 0 that impactsabout 60,000 women in the US each year. These women are at elevated risk(25%) of having another BC event. Despite the excellent prognosis and98% survival, there are some suggestions that women including thosebelow age 40, African American women and those with estrogenreceptor-negative (ER^(neg)) DCIS display a 7-15% chance of dying fromsubsequent BC probably because disseminated cancer cells (DCC) thatescape prior to clinical detection of invasive breast cancer (IBC).Approximately 33-50% of high grade DCIS lesions express HER2 protein andanother one third display modest HER2 expression. We have shown thesewomen with HER2 DCIS have significantly greater likelihood of having anIBC component identified in their DCIS usually T1a/b (T1a are tumorsunder 5 mm T1b are tumors that are 5 mm-1 cm (see, e.g., American JointCommittee on Cancer (AJCC) Staging Manual-8^(th) Edition, Amin, M. B.,et al. Eds., Springer Nature (2017)) and increased risk of ipsilateralbreast recurrence. In patients with T1b IBC the risk of subsequentmortality increases to 20-30% thus most of these women are offeredadjuvant chemotherapy with trastuzumab to decrease risk. Althougheffective, even weekly paclitaxel and trastuzumab can result inneurologic, cardiac, cognitive as well as other morbidities. Many ofthese patients presenting with larger areas of DCIS mixed with T1a/b IBCand receive even more intense chemotherapy regimens like PTCH becausethe T stage of IBC can be difficult to discern. Many also requiremastectomies because of the large areas of DCIS that do not alwaysrespond to neoadjuvant therapy. If these tumors are also estrogenreceptor positive, patients are also treated with an additional 5 yearsof anti-estrogen further enhancing morbidity. To summarize, some HER2DCIS patients are potentially undertreated and left with elevated riskof subsequent breast events even slightly increased mortality and theT1a/b patients can not infrequently be over-treated. Since existingtargeted strategies are less effective, there is a need for attractiveimmunotherapeutic strategies.

SUMMARY

Disclosed are methods and compositions related to novel combinationtherapies comprising oncodriver pulsed dendritic cells andimmunoregulator molecules inhibitors.

In one aspect, disclosed herein are anti-cancer combination therapiescomprising at least one dendritic cell pulsed with an oncodriver (suchas, for example, human epidermal growth factor receptor (HER) 2(HER2))and at least one inhibitor of an immunoregulatory molecule (such as, forexample, Semaphorin (SEMA) 4D (SEMA4D), or VEGF); wherein theimmunoregulatory molecule being inhibited effects the vasculature of atumor.

In one aspect, disclosed herein are methods of treating, preventing,reducing, and/or inhibiting a cancer (such as, for example, breastcancer (including triple negative breast cancer, metastatic breastcancer (MBC), ductal carcinoma in situ (DCIS), and invasive breastcancer (IBC)), melanoma, colorectal cancer, pancreatic cancer, prostatecancer, bladder cancer, ovarian cancer, and stomach cancer, andincluding primary and distant tumors) in a subject comprisingadministering the anti-cancer combination therapy of any precedingaspect. Thus, in one aspect, disclosed herein are methods of treating acancer in a subject comprising administering to the subject at least onedendritic cell pulsed with an oncodriver (such as, for example, humanepidermal growth factor receptor (HER) 2 (HER2)) and at least oneinhibitor of an immunoregulatory molecule (such as, for example,Semaphorin (SEMA) 4D (SEMA4D), or VEGF); wherein the immunoregulatorymolecule being inhibited effects the vasculature of a tumor.

Also disclosed are anti-cancer combination therapies methods treating,preventing, reducing, and/or inhibiting a cancer of any precedingaspect; wherein the at least one immunoregulatory molecule inhibitor isadministered systemically and/or the oncodriver pulsed dendritic cell isadministered intratumorally.

In one aspect, also disclosed herein are anti-cancer combinationtherapies methods treating, preventing, reducing, and/or inhibiting acancer of any preceding aspect; wherein the oncodriver pulsed dendriticcell is activated with IL-12 prior to administration.

Also disclosed herein are anti-cancer combination therapies methodstreating, preventing, reducing, and/or inhibiting a cancer of anypreceding aspect; wherein the at least one immunoregulator moleculeinhibitor comprises an antibody or functional fragment thereof whichbinds to SEMA4D (also referred to herein as CD100) such as, for example,the anti-SEMA4D antibodies Mab 67 or VX15/2503 (Pepinemab). See, e.g.,U.S. Pat. No. 8,496,938, incorporated herein by reference.

In one aspect, also disclosed herein are anti-cancer combinationtherapies methods treating, preventing, reducing, and/or inhibiting acancer of any preceding aspect; wherein the dendritic cells are removedfrom the subject and pulsed with oncodriver ex vivo.

Also disclosed herein are anti-cancer combination therapies methodstreating, preventing, reducing, and/or inhibiting a cancer of anypreceding aspect; wherein the pulsed dendritic cells are administered atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4,5, or 6 months prior to administration of the at least oneimmunoregulatory molecule inhibitor; are administered concurrently withthe at least one immunoregulatory molecule inhibitor; or wherein the atleast one immunoregulatory molecule inhibitor is administered at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, or6 months prior to administration of the pulsed dendritic cells.

In one aspect, disclosed herein are anti-cancer combination therapiesmethods treating, preventing, reducing, and/or inhibiting a cancer ofany preceding aspect; wherein the at least one pulsed dendritic cell isadministered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day or at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times per week for at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 weeks.

Also disclosed herein are anti-cancer combination therapies methodstreating, preventing, reducing, and/or inhibiting a cancer of anypreceding aspect; wherein the at least one immunoregulatory moleculeinhibitor is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day orat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times per weekfor at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 weeks.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description illustrate the disclosed compositions and methods.

FIGS. 1A, 1B, 1C, and 1D show progressive loss of anti-oncodriver Th1responses with advancing oncodriver-expressing breast disease, andassociation of anti-oncodriver immunity with improved clinical outcomes.FIG. 1A shows that loss of HER-2 Th1 response with HER-2 positivedisease progression; FIG. 1B shows that vaccine, but not standardtherapy helps restore anti-HER-2 Th1 immunity; FIG. 1C shows that Th1immunity to HER-2 predicts clinical response to standard therapy; andFIG. 1D shows that HER-2 Th1 responsivity predicts disease-free survivalafter standard therapy. Acronyms: DCIS: ductal carcinoma in situ; IBC:invasive breast cancer; Tx: treatment; pCR: pathologic completeresponse; ER: estrogen receptor; TNBC: triple-negative breast cancer.

FIGS. 2A, 2B, and 2C show short-course anti-estrogen therapy concurrentwith DC1 vaccination improves pCR rate (FIG. 2A) and anti-HER2 Th1immunity (FIG. 2B) for subjects with hormone-dependent (ER-positive)disease, and that pathologic complete response (pCR) predicts long-termfreedom from subsequent breast events (SBE) for all vaccinated subjects(FIG. 2C).

FIG. 3A-3G show Intratumoral DC1 in combination with anti-SEMA4Dantibody induced tumor regression in HER2 positive TUBO model. FIG. 3A:SEMA4D expression in TUBO cells as measured by immunohistochemistry,compared to an IgG isotype control antibody; FIG. 3B: Intratumoral classII HER2 peptide pulsed DC1 intratumoral injection in combination withanti-SEMA4D antibody given intraperitoneally in single tumor model (FIG.3B1) and survival curve (FIG. 3B2); FIG. 3C: Efficacy of intratumoralDC1 in combination with anti-SEMA4D in bilateral model (FIG. 3C1 andFIG. 3C2); TUBO bearing mice were treated with either with unpulsed orclass II HER2 peptide pulsed activated DC1 alone or in combination withanti-SEMA4D antibody; FIG. 3D: IHC staining of SEMA4D in DCIS and IBCpatients, the numbers indicate the number of patients with positiveSEMA4D staining; FIG. 3E: Myeloid-derived suppressor cell (MDSC)infiltration per mg of tumor from treated with DC1 alone, anti-SEMA4Dalone or in combination; FIG. 3F: CD4 T cell infiltration per mg oftumor; FIG. 3G: CD4 T cell infiltration (absolute number) in lymph node.

FIG. 4A, 4B, and 4C show that T cells from HER2-DC1+aSema4D treated micewere superior in proliferation, function and specificity compared to Tcells from HER2-DC1 alone vaccinated mice. FIG. 4A: Fold expansion afterthree weeks of in vitro CD4 T cell expansion. CD4+ T cells were isolatedfrom isolated splenocytes from vaccinated mice using EASYSEP™ (StemcellTechnologies) and co-cultured with HER2 peptide pulsed DC1 followed byexpansion with IL-2 and IL-7 for three weeks; FIG. 4B: Expanded T cellsfrom DC1+ anti-SEMA4D treated mice were more antigen specific for theHER2/neu peptide antigens p5, p435, and p1209 compared to T cells fromDC1 vaccinated mice; FIG. 4C: Cumulative IFN-γ response of expanded Tcells.

FIGS. 5A, 5B, and 5C show detection of DCC in neu Transgenic mice. FIG.5A: Flow staining of DCC in bone marrow (BM) by cytokeratin 8/18 andHER2 expression; FIG. 5B: Immunofluorescence staining of HER2,cytokeratin and ki67; FIG. 5C: Detection of DCC in different organs ofneu T mice.

FIGS. 6A, 6B, 6C, and 6D show HER2 peptide-DC1 vaccine prevents mammarytumor development, induce senescence and eliminate DCC in NeuT mice.FIG. 6A: Immunofluorescence staining of bone marrow DCC from control andDC1 vaccinated neu T mice; FIG. 6B: Detectable tumor mass in mammaryglands using ultrasound; FIG. 6C: Percent DCC in bone marrow measuredusing flow cytometry; FIG. 6D: Detection of senescent cells using β-galassay. Representative images after the β-gal staining.

FIGS. 7A and 7B show that HER2 peptide pulsed DC1 vaccine andanti-SEMA4D treatment induced infiltration of B cells in the mammaryglands and CD4+ T cell infiltration in bone marrow of NeuT mice. FIG.7A: 8 weeks old NeuT mice were treated with HER2 peptide pulsed DC1vaccine (two injections per week for three weeks) or anti-SEMA4Dantibody. On week 16, mice were sacrificed, and mammary glands werecollected. The single cell suspension of mammary glands was prepared andstained for CD19 positive B cells then analyzed by flow cytometry.Increased levels of CDa19+ B cells were observed in DC1 vaccinated miceand anti-SEMA4D treated mice compared to untreated control; FIG. 7B: Thebone marrow cells derived from the control and DC1 vaccinated NeuT micewere stained for CD4 and CD8 T cell markers and analyzed by flowcytometry. Increased numbers of CD4T cells and CD8 T cells were observedin the bone marrow of DC1 vaccinated NeuT mice compared to untreatedcontrol mice.

FIGS. 8A, 8B, 8C, 8D, and 8E show Dual Blockade of HER2 and HER3 incombination with Th1 cytokine mediates tumor senescence and apoptosis inSK-BR-3 breast cancer cells. FIG. 8A: Dual blockade of HER2/HER3 inSK-BR-3 cells combined with Th1 cytokines TNF-α and IFN-γ enhance thenumber of senescent cells, higher SA-β-gal staining was observed incells; FIG. 8B: SK-BR-3 cells untreated (1), treated with TNF-α andIFN-γ (2), or treated with trastuzumab (Herceptin, H (TZm)) andpertuzumab (Per) (3), or treated with TNF-α, IFN-γ and TZm and Per (4);FIG. 8C: Western blot analysis of SK-BR-3 cells treated with Th1cytokines in combination with Tzm and Per induced Cyclin-dependentkinase 4 inhibitor B, also known as p15^(INK4b) and cleaved caspase-3expression (treatments numbered as in panel B); and FIG. 8D and FIG. 8E:Apoptosis by Annexin V/PI staining (treatments numbered as in panels D&E).

FIGS. 9A, 9B, and 9C show Th1 cytokines TNF-α and IFN-γ inducessenescence and apoptosis in trastuzumab and pertuzumab resistant breastcancer cells. FIG. 9A: HCC-1419 and JIMT-1 cells; 1) untreated; 2)treated with TNF-α and IFN-γ; 3) treated with TZm and Per, or 4) treatedwith TNF-α, IFN-γ, Tzm, and Per. FIG. 9A. % of SA-β-gal-positive cells;FIG. 9B: Western blots analysis of p15^(INKb) and cleaved caspase-3expression of HCC-1419 post treatment; and FIG. 9C: Western blotanalysis of JIMT-1 cells. Vinculin was used as a control.

FIGS. 10A and 10B show HER2-specific CD4⁺ Th1-mediated senescence andapoptosis of HER2-ovexpressing human breast cancer cells. FIG. 10A:SK-BR-3 cells co-cultured with CD4⁺ T-cells alone (CD4⁺ only (1)), CD4⁺T-cells+HER2 peptide-pulsed immature dendritic cells (CD4⁺ IDC H (2)),CD4⁺ T-cells+HER2 peptide-pulsed mature dendritic cells (CD4⁺ DC H (3)),or CD4⁺ DC H with trastuzumab (Tzm) and pertuzumab (Per) (4), or CD4⁺T-cells+irrelevant peptide-pulsed mature dendritic cells (BRAF (CD4⁺ DCB) (5); or survivin (CD4⁺ DC S)(6)), with Tzm and Per. FIG. 10B: Westernblot analysis of tumor cells showed increase in p15^(INK4b) and cleavedcaspase-3 expression suggests induced senescence and apoptosis,respectively, when co-cultured with the DC H/CD4⁺ T-cells in presence ofTzm and Per, but not from DC B, DC S and iDC H groups. Vinculin was usedas loading control

FIG. 11 shows IFN-γ administered subcutaneously twice weekly with weeklyTaxol and standard dose trastuzumab and pertuzumab in patients withfirst line metastatic breast cancer was safe and resulted in diseasestabilization of partial responses.

FIGS. 12A, 12B, 12C, and 12D show immunohistochemical staining oflymphocyte infiltration before and after DC1 vaccine in DCIS. FIG. 12Aand FIG. 12B show areas of dense lymphocyte infiltrate; FIG. 12C andFIG. 12D show areas with little or no response.

FIG. 13 shows accumulation of lymphocytes pre and post DC1 vaccinationin DCIS. CD4, CD8 and CD20 infiltration is shown.

FIG. 14 shows Lymphocytes infiltration in DCIS ducts after DC1vaccination.

FIGS. 15A and 15B show combination therapy with intratumoral DC1 andanti-SEMA4D improves tumor vascularity. FIG. 15A: Slope of the DCE-MRIcurves calculated after iv administration of Gadavist (agadolinium-based MRI contrast agent, 0.2 mmol/kg). Despite tumor volume,combo treatment shows a smaller slope which indicates less vesselleakage; FIG. 15B: DCE-MRI curves of TUBO tumors showing thatcombination therapy has smaller DCE curve. Data is represented asrelative value (respect to the first point of the curve) to show areliable comparison among the tumors.

FIG. 16 shows CEST MRI (tumor pH) map of a TUBO tumor treated withanti-SEMA4D. From right to left: T2-weighted image representing the ROI,pH map the insertion shows the pH mean value and its StandardDeviation), and histogram representing pH values of all pixelscalculated.

FIGS. 17A-F show that CD4+ T cells are required for anti-SEMA4Dactivity. FIG. 17A: mean tumor size (mm²) in a murine Her2 TUBO breastcancer model over time following treatment with Her2DC1, anti-SEMA4D,HER2 DC1 plus anti-SEMA4D, or Control IgG. FIG. 17B: percent survival ofHer2 TUBO tumor-bearing mice following treatment with Her2DC1,anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG. FIG. 17C: meantumor size (mm²) in CD4-depleted Her2 TUBO tumor-bearing mice over timefollowing treatment with Her2DC1, anti-SEMA4D, HER2 DC1 plusanti-SEMA4D, or no treatment. FIG. 17D: percent survival of CD4-depletedHer2 TUBO tumor-bearing mice following treatment with Her2DC1,anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or no treatment. FIG. 17E: tumorsize (mm²) in individual Her2 TUBO tumor-bearing mice over timefollowing treatment with Her2DC1, anti-SEMA4D, HER2 DC1 plusanti-SEMA4D, or Control IgG. Complete tumor regressions are observed incontrol Balb/c mice following treatment with anti-SEMA4D and combinationof anti-SEMA4D plus Her2DC1. FIG. 17F: tumor size (mm²) in individualCD4-depleted Her2 TUBO tumor-bearing mice over time following treatmentwith Her2DC1, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG.(17E, F: Tumor growth curves for each mouse are shown; CR=complete tumorregression; tumor volume <50 mm2.) (*p<0.05, **p<0.01)

FIGS. 18A-C show that Fc Receptor gamma (FcRγ) is required for completetumor regression following treatment with combination therapy. FIG. 18A:mean tumor size (mm²) in BALB/C.129P2(B6)-Fcer1g^(tm1Rav) N12tumor-bearing mice treated with HER2DC, anti-SEMA4D, HER2DC plusanti-SEMA4D; or no treatment. FIG. 18B: percent survival ofC.129P2(B6)-Fcer1g^(tm1Rav) N12 tumor-bearing mice treated with HER2DC,anti-SEMA4D, HER2DC plus anti-SEMA4D; or no treatment. FIG. 18C: Tumorgrowth curves for each mouse are shown. No complete tumor regressionsare observed in FcRγ-deficient mice. (*p<0.05, **p<0.01)

FIG. 19 shows that Interferon gamma (IFN-γ) is required for anti-tumoractivity of DC1, anti-SEMA4D, and combination therapy. Mean tumor size(mm²) in Balb/C IFN-γ knock out (KO) (C.129S7 (B6)-IFNg^(tm1Ts)/J(IFN-γ^(KO), Jackson Laboratories) mice that carried tumors generatedfrom murine Her2 TUBO breast cancer cells is shown over time followingtreatment with Her2DC1, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, orControl IgG.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

DEFINITIONS

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims that follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

A “decrease” can refer to any change that results in a smaller amount ofa symptom, disease, composition, condition, or activity. A substance isalso understood to decrease the genetic output of a gene when thegenetic output of the gene product with the substance is less relativeto the output of the gene product without the substance. Also forexample, a decrease can be a change in the symptoms of a disorder suchthat the symptoms are less than previously observed. A decrease can beany individual, median, or average decrease in a condition, symptom,activity, composition in a statistically significant amount. Thus, thedecrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long asthe decrease is statistically significant.

“Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity,response, condition, disease, or other biological parameter. This caninclude but is not limited to the complete ablation of the activity,response, condition, or disease. This may also include, for example, a10% reduction in the activity, response, condition, or disease ascompared to the native or control level. Thus, the reduction can be a10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction inbetween as compared to native or control levels.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant lowering of an event or characteristic (e.g.,tumor growth). It is understood that this is typically in relation tosome standard or expected value, in other words it is relative, but thatit is not always necessary for the standard or relative value to bereferred to. For example, “reduces tumor growth” means reducing the rateof growth of a tumor relative to a standard or a control.

By “prevent” or other forms of the word, such as “preventing” or“prevention,” is meant to stop a particular event or characteristic, tostabilize or delay the development or progression of a particular eventor characteristic, or to minimize the chances that a particular event orcharacteristic will occur. Prevent does not require comparison to acontrol as it is typically more absolute than, for example, reduce. Asused herein, something could be reduced but not prevented, but somethingthat is reduced could also be prevented. Likewise, something could beprevented but not reduced, but something that is prevented could also bereduced. It is understood that where reduce or prevent are used, unlessspecifically indicated otherwise, the use of the other word is alsoexpressly disclosed.

“Biocompatible” generally refers to a material and any metabolites ordegradation products thereof that are generally non-toxic to therecipient and do not cause significant adverse effects to the subject.

“Comprising” is intended to mean that the compositions, methods, etc.include the recited elements, but do not exclude others. “Consistingessentially of” when used to define compositions and methods, shall meanincluding the recited elements, but excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions provided and/or claimedin this disclosure. Embodiments defined by each of these transitionterms are within the scope of this disclosure.

A “control” is an alternative subject or sample used in an experimentfor comparison purposes. A control can be “positive” or “negative.”

“Effective amount” of an agent refers to a sufficient amount of an agentto provide a desired effect. The amount of agent that is “effective”will vary from subject to subject, depending on many factors such as theage and general condition of the subject, the particular agent oragents, and the like. Thus, it is not always possible to specify aquantified “effective amount.” However, an appropriate “effectiveamount” in any subject case may be determined by one of ordinary skillin the art using routine experimentation. Also, as used herein, andunless specifically stated otherwise, an “effective amount” of an agentcan also refer to an amount covering both therapeutically effectiveamounts and prophylactically effective amounts. An “effective amount” ofan agent necessary to achieve a therapeutic effect may vary according tofactors such as the age, sex, and weight of the subject. Dosage regimenscan be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

A “pharmaceutically acceptable” component can refer to a component thatis not biologically or otherwise undesirable, i.e., the component may beincorporated into a pharmaceutical formulation provided by thedisclosure and administered to a subject as described herein withoutcausing significant undesirable biological effects or interacting in adeleterious manner with any of the other components of the formulationin which it is contained. When used in reference to administration to ahuman, the term generally implies the component has met the requiredstandards of toxicological and manufacturing testing or that it isincluded on the Inactive Ingredient Guide prepared by the U.S. Food andDrug Administration.

“Pharmaceutically acceptable carrier” (sometimes referred to as a“carrier”) means a carrier or excipient that is useful in preparing apharmaceutical or therapeutic composition that is generally safe andnon-toxic and includes a carrier that is acceptable for veterinaryand/or human pharmaceutical or therapeutic use. The terms “carrier” or“pharmaceutically acceptable carrier” can include, but are not limitedto, phosphate buffered saline solution, water, emulsions (such as anoil/water or water/oil emulsion) and/or various types of wetting agents.As used herein, the term “carrier” encompasses, but is not limited to,any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer,lipid, stabilizer, or other material well known in the art for use inpharmaceutical formulations and as described further herein.

“Pharmacologically active” (or simply “active”), as in a“pharmacologically active” derivative or analog, can refer to aderivative or analog (e.g., a salt, ester, amide, conjugate, metabolite,isomer, fragment, etc.) having the same type of pharmacological activityas the parent compound and approximately equivalent in degree.

“Polymer” refers to a relatively high molecular weight organic compound,natural or synthetic, whose structure can be represented by a repeatedsmall unit, the monomer. Non-limiting examples of polymers includepolyethylene, rubber, cellulose. Synthetic polymers are typically formedby addition or condensation polymerization of monomers. The term“copolymer” refers to a polymer formed from two or more differentrepeating units (monomer residues). By way of example and withoutlimitation, a copolymer can be an alternating copolymer, a randomcopolymer, a block copolymer, or a graft copolymer. It is alsocontemplated that, in certain aspects, various block segments of a blockcopolymer can themselves comprise copolymers. The term “polymer”encompasses all forms of polymers including, but not limited to, naturalpolymers, synthetic polymers, homopolymers, heteropolymers orcopolymers, addition polymers, etc.

A “binding molecule” or “antigen binding molecule” (e.g., an antibody orantigen-binding fragment thereof) as provided herein refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant In one embodiment, the binding molecule specifically bindsto an immunoregulator molecule (such as for example, a transmembraneSEMA4D (CD100) polypeptide of about 150 kDa or a soluble SEMA4Dpolypeptide of about 120 kDa). In another embodiment, a binding moleculeis an antibody or an antigen binding fragment thereof, e.g., MAb 67 orpepinemab.

“Therapeutic agent” refers to any composition that has a beneficialbiological effect. Beneficial biological effects include boththerapeutic effects, e.g., treatment of a disorder or other undesirablephysiological condition, and prophylactic effects, e.g., prevention of adisorder or other undesirable physiological condition (e.g., anon-immunogenic cancer). The terms also encompass pharmaceuticallyacceptable, pharmacologically active derivatives of beneficial agentsspecifically mentioned herein, including, but not limited to, salts,esters, amides, proagents, active metabolites, isomers, fragments,analogs, and the like. When the terms “therapeutic agent” is used, then,or when a particular agent is specifically identified, it is to beunderstood that the term includes the agent per se as well aspharmaceutically acceptable, pharmacologically active salts, esters,amides, proagents, conjugates, active metabolites, isomers, fragments,analogs, etc.

“Therapeutically effective amount” or “therapeutically effective dose”of a composition (e.g. a composition comprising an agent) refers to anamount that is effective to achieve a desired therapeutic result. Insome embodiments, a desired therapeutic result is the control of type Idiabetes. In some embodiments, a desired therapeutic result is thecontrol of obesity. Therapeutically effective amounts of a giventherapeutic agent will typically vary with respect to factors such asthe type and severity of the disorder or disease being treated and theage, gender, and weight of the subject. The term can also refer to anamount of a therapeutic agent, or a rate of delivery of a therapeuticagent (e.g., amount over time), effective to facilitate a desiredtherapeutic effect, such as pain relief. The precise desired therapeuticeffect will vary according to the condition to be treated, the toleranceof the subject, the agent and/or agent formulation to be administered(e.g., the potency of the therapeutic agent, the concentration of agentin the formulation, and the like), and a variety of other factors thatare appreciated by those of ordinary skill in the art. In someinstances, a desired biological or medical response is achievedfollowing administration of multiple dosages of the composition to thesubject over a period of days, weeks, or years.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

Compositions

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular immunoregulator molecule inhibitor oroncodriver pulsed dendritic cell is disclosed and discussed and a numberof modifications that can be made to a number of molecules including theimmunoregulator molecule inhibitor or oncodriver pulsed dendritic cellare discussed, specifically contemplated is each and every combinationand permutation of immunoregulator molecule inhibitor or oncodriverpulsed dendritic cell and the modifications that are possible unlessspecifically indicated to the contrary. Thus, if a class of molecules A,B, and C are disclosed as well as a class of molecules D, E, and F andan example of a combination molecule, A-D is disclosed, then even ifeach is not individually recited each is individually and collectivelycontemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E,and C-F are considered disclosed. Likewise, any subset or combination ofthese is also disclosed. Thus, for example, the sub-group of A-E, B-F,and C-E would be considered disclosed. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed compositions. Thus, if thereare a variety of additional steps that can be performed it is understoodthat each of these additional steps can be performed with any specificembodiment or combination of embodiments of the disclosed methods.

Patients presenting with DCIS in general have excellent prognosishowever those presenting at age <40, African American females, andER^(neg) DCIS have modestly increased risk of dying of subsequent BCthat neither surgery nor radiation appears to prevent. A second problemis that many young patients also present with larger regions of HER2expressing DCIS that contains areas of T1a/T1b invasion. These patientsare typically either treated with mastectomy because of the size of areaof calcifications or treated with strong neoadjuvant chemotherapyregimens of carboplatin, taxotere with trastuzumab and pertuzumab (PTCH)or taxol and trastuzumab (TH) with good survival but to suffer the longterm consequences of extensive surgery, radiation and chemotherapy. In aday of personalized medicine these patients need more personalizedeffective therapy that both reduces the odds of subsequent BC mortalityand at the same time reduces the overtreatment they receive fromchemotherapy, a year of trastuzumab, radiation and often mastectomies.Patients with metastatic breast cancer (MBC) are in desperate need ofnew immunotherapies to reduce mortality especially those that becomeresistant to targeted therapies.

HER-2/neu over-expression plays a critical role in breast cancer (BC)development and its expression in ductal carcinoma in situ (DCIS) isassociated with development of invasive BC (IBC). There is a progressiveloss of the systemic anti-HER2 Th1 immune response in HER2 positive DCISand invasive BC patients. Administration of class II HER2 peptide-pulsedType I polarized dendritic cell vaccine (HER2-DC1) partially restoredanti-HER2 Th1 immune responses with about 30% pathologic completeresponse rate (pCR) in DCIS. There is opportunity to improve the immuneresponse and clinical activity in patients with early HER2 BC.Semaphorin 4D (SEMA4D) is a family of soluble and transmembrane proteinsthat are essential for tissue and organ development and are involved inimmune regulation. Overexpression of SEMA4D correlates with poorprognosis and tumor progression in various cancers. In this study, amurine anti-SEMA4D monoclonal antibody (provided by Vaccinex) incombination with DC1 vaccine was investigated to enhance anti-tumorimmune response in a preclinical model of HER2 positive TUBO breastcancer.

In one aspect, disclosed herein are anti-cancer combination therapiescomprising at least one dendritic cell pulsed with an oncodriver (suchas, for example, human epidermal growth factor receptor HER2, and atleast one inhibitor of an immunoregulatory molecule (such as, forexample, Semaphorin (SEMA) 4D (SEMA4D), or VEGF). In certain nonlimitingaspects, the immunoregulatory molecule being inhibited can affect thevasculature of a tumor.

It is understood and herein contemplated that the disclosed anti-cancercombination therapies can be used to treat, prevent, reduce, and/orinhibit any disease where uncontrolled cellular proliferation occurssuch as cancers including primary and distant tumors. A non-limitinglist of different types of cancers is as follows: lymphomas (Hodgkinsand non-Hodgkins), leukemias, carcinomas, carcinomas of solid tissues,squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, high gradegliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas,melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphomas orsarcomas, metastatic cancers, or cancers in general.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, lung cancers suchas small cell lung cancer and non-small cell lung cancer,neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer,melanoma, squamous cell carcinomas of the mouth, throat, larynx, andlung, cervical cancer, cervical carcinoma, breast cancer (includingtriple negative breast cancer, metastatic breast cancer (MBC), ductalcarcinoma in situ (DCIS), and invasive breast cancer (IBC)), andepithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer,esophageal carcinoma, head and neck carcinoma, large bowel cancer,hematopoietic cancers; testicular cancer; colorectal cancer, prostaticcancer, or pancreatic cancer. Thus, in one aspect, disclosed herein aremethods of treating, preventing, reducing, and/or inhibiting a cancer(such as, for example, breast cancer (including triple negative breastcancer, metastatic breast cancer (MBC), ductal carcinoma in situ (DCIS),and invasive breast cancer (IBC)), melanoma, colorectal cancer,pancreatic cancer, and prostate cancer and including primary and distanttumors) in a subject comprising administering the anti-cancercombination therapy of any preceding aspect. For example, disclosedherein are methods of treating, preventing, reducing, and/or inhibitinga cancer in a subject comprising administering to the subject at leastone dendritic cell pulsed with an oncodriver (such as, for example,human epidermal growth factor receptor (HER) HER2, and at least oneinhibitor of an immunoregulatory molecule (such as, for example,Semaphorin (SEMA) 4D (SEMA4D), or VEGF). In certain nonlimiting aspects,the immunoregulatory molecule being inhibited can affect the vasculatureof a tumor.

In one aspect, it is understood that the disclosed methods andanti-cancer combination therapies comprise inhibitor of animmunoregulatory molecules that have both an immunoregulatory effect,and in certain nonlimiting aspects can also affect the vasculature of atumor. It is understood and herein contemplated that said inhibitors cancomprise any small molecule, peptide, protein, antibody (including anyfunctional fragments of an antibody or other binding molecule), and/orfunctional nucleic acid (siRNA, RNA, aptamer) that inhibits theimmunoregulatory and/or vascular activity of the immunoregulatorymolecule. In one aspect, the inhibitor of an immunoregulatory moleculecomprises the SEMA4D inhibitor pepinemab (an anti-SEMA4D antibody)

Antibodies Antibodies Generally

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, aslong as they are chosen for their ability to interact with animmunoregulatory molecule (such as, for example, Semaphorin (SEMA) 4D(SEMA4D), or VEGF) such that the immunoregulator molecule is inhibitedfrom its immunoregulatory activity and/or its effects on the vasculatureof a tumor. The antibodies can be tested for their desired activityusing the in vitro assays described herein, or by analogous methods,after which in vivo therapeutic and/or prophylactic activities can betested according to known clinical testing methods. Native antibodiesare usually heterotetrameric glycoproteins, composed of two identicallight (L) chains and two identical heavy (H) chains. Typically, eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies between the heavy chainsof different immunoglobulin isotypes. Each heavy and light chain alsohas regularly spaced intrachain disulfide bridges. Each heavy chain hasat one end a variable domain (V(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains. The lightchains of antibodies from any vertebrate species can be assigned to oneof two clearly distinct types, called kappa (k) and lambda (l), based onthe amino acid sequences of their constant domains. Depending on theamino acid sequence of the constant domain of their heavy chains,immunoglobulin s can be assigned to different classes. There are fivemajor classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled inthe art would recognize the comparable classes for mouse. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively.

The term “variable” is used herein to describe certain portions of thevariable domains that differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a b-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the b-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat E. A.et al., “Sequences of Proteins of Immunological Interest,” NationalInstitutes of Health, Bethesda, Md. (1987)). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein (such as, for example theanti-SEMA4D antibody pepinemab), the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers are not identical. For example, each monomermay comprise a different target binding site, forming, for example, abispecific antibody.

The heavy chain portions of a binding molecule for use in the diagnosticand treatment methods disclosed herein may be derived from differentimmunoglobulin molecules. For example, a heavy chain portion of apolypeptide may comprise a C_(H1) domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain, e.g., a kappa orlambda light chain. Preferably, the light chain portion comprises atleast one of a VL or CL domain.

Anti-SEMA4D antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein can be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide disclosed herein (e.g., SEMA4D) that they recognize orspecifically bind. The portion of a target polypeptide that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant ” A target polypeptide maycomprise a single epitope, but typically comprises at least twoepitopes, and can include any number of epitopes, depending on the size,conformation, and type of antigen. Furthermore, it should be noted thatan “epitope” on a target polypeptide may be or may includenon-polypeptide elements, e.g., an epitope may include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. A peptide or polypeptide epitope recognized by anti-SEMA4Dantibodies may contain a sequence of at least 4, at least 5, at least 6,at least 7, more preferably at least 8, at least 9, at least 10, atleast 15, at least 20, at least 25, or between about 15 to about 30contiguous or non-contiguous amino acids of SEMA4D.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity.

The disclosed monoclonal antibodies can be made using any procedurewhich produces mono clonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The monoclonal antibodies may also be made by recombinant DNA methods.DNA encoding the disclosed monoclonal antibodies can be readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). Libraries ofantibodies or active antibody fragments can also be generated andscreened using phage display techniques, e.g., as described in U.S. Pat.No. 5,804,440 to Burton et al. and U.S. Pat. No. 6,096,441 to Barbas etal.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

As used herein, the term “antibody or fragments thereof” encompasseschimeric antibodies and hybrid antibodies, with dual or multiple antigenor epitope specificities, and fragments, such as F(ab′)2, Fab′, Fab, Fd,Fv, scFv, disulfide-linked Fvs (sdFv), fragments comprising either orboth V_(H) or V_(L) domain, and the like, including hybrid fragments.Thus, fragments of the antibodies that retain the ability to bind theirspecific antigens are provided. For example, fragments of antibodieswhich maintain immunoregulatory molecule (such as, for example,Semaphorin (SEMA) 4D (SEMA4D), or VEGF) binding activity are includedwithin the meaning of the term “antibody or fragment thereof.” Suchantibodies and fragments can be made by techniques known in the art andcan be screened for specificity and activity according to the methodsset forth in the Examples and in general methods for producingantibodies and screening antibodies for specificity and activity (SeeHarlow and Lane. Antibodies, A Laboratory Manual. Cold Spring HarborPublications, New York, (1988)).

Also included within the meaning of “antibody or fragments thereof” areconjugates of antibody fragments and antigen binding proteins (singlechain antibodies).

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof.

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse.

Human Antibodies

The disclosed human antibodies can be prepared using any technique. Thedisclosed human antibodies can also be obtained from transgenic animals.For example, transgenic, mutant mice that are capable of producing afull repertoire of human antibodies, in response to immunization, havebeen described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, thehomozygous deletion of the antibody heavy chain joining region (J(H))gene in these chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production, and the successfultransfer of the human germ-line antibody gene array into such germ-linemutant mice results in the production of human antibodies upon antigenchallenge. Antibodies having the desired activity are selected usingEnv-CD4-co-receptor complexes as described herein.

Humanized Antibodies

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as ansFv, Fv, Fab, Fab′, F(ab′)2, or other antigen-binding portion of anantibody) which contains a portion of an antigen binding site from anon-human (donor) antibody integrated into the framework of a human(recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5, 939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

Anti-SEMA4D Antibodies

In one aspect, the inhibitor of an immunoregulatory molecule can be theSEMA4D inhibitor pepinemab (an anti-SEMA4D antibody) such as thosedescribed in U.S. Pat. Nos. 8,496,938, 8,816,058, 9,605,055, and9,676,840, patents which are incorporated herein by reference for theirteachings of anti-SEMA4D (anti-DC100) antibodies. Anti- SEMA4Dmonoclonal antibodies have been developed to neutralize SEMA4D,including MAb 67, MAb 2503, and MAb 76.

Antibodies that bind SEMA4D have been described the art. See, forexample, US Publ. No. 2008/0219971 A1, International Patent ApplicationWO 93/14125 and Herold et al., Int. Immunol. 7(1): 1-8 (1995), each ofwhich is herein incorporated in its entirety by reference.

Anti-SEMA4D antibodies or antigen-binding fragments, variants, orderivatives thereof can include, e.g., MAb 2503, MAb 67, or MAb 76. Incertain embodiments the anti- SEMA4D antibodies bind human, murine, orboth human and murine SEMA4D. In other embodiments, the anti-SEMA4Dantibodies block SEMA4D binding to its receptor, e.g., Plexin-B1 orPlexin-B2.

It is understood and herein contemplated that the disclosed anti-cancercombination therapies and methods of treating, inhibiting, reducing,and/or preventing a cancer using said anti-cancer combination therapiescan comprise more than one immunoregulator molecule inhibitor and morethan one population of pulsed dendritic cells with each population ofpulsed dendritic cells being pulsed with the same or differentoncodrivers.

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. In one aspect, the subject can be human, non-humanprimate, bovine, equine, porcine, canine, or feline. The subject canalso be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, thesubject can be a human or veterinary patient. The term “patient” refersto a subject under the treatment of a clinician, e.g., physician.

The term “therapeutically effective” refers to the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

In one aspect, it is understood and herein contemplated that the pulseddendritic cells can be activated prior to administration as well asprior to being pulsed with the oncodriver. Activation of the dendriticcells (DC1) can be achieved by contacting the cells with IFN-γ, TNFα,CD40, IL21, and/or IL-12. In one aspect, it is further understood thatthe subject's own dendritic cells can be removed and pulsed ex vivo andtransferred back to the subject for use in the disclosed anti-cancercombination therapies for treating, preventing, reducing, and/orinhibiting a cancer.

It is understood and herein contemplated that the disclosed anti-cancercombination therapies can be administered via any route determined to beappropriate by the attending physician. Administration” to a subjectincludes any route of introducing or delivering to a subject an agenteither locally and/or systemically. Administration can be carried out byany suitable route, including oral, topical, intravenous, subcutaneous,transcutaneous, transdermal, intramuscular, intra-joint, parenteral,intra-arteriole, intradermal, intraventricular, intracranial,intraperitoneal, intralesional, intranasal, rectal, vaginal, byinhalation, via an implanted reservoir, parenteral (e.g., subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intratumoral, intrasternal, intrathecal, intraperitoneal, intrahepatic,intralesional, and intracranial injections or infusion techniques), andthe like. “Concurrent administration”, “administration in combination”,“simultaneous administration” or “administered simultaneously” as usedherein, means that the compounds are administered at the same point intime or essentially immediately following one another. In the lattercase, the two compounds are administered at times sufficiently closethat the results observed are indistinguishable from those achieved whenthe compounds are administered at the same point in time. “Systemicadministration” refers to the introducing or delivering to a subject anagent via a route which introduces or delivers the agent to extensiveareas of the subject's body (e.g. greater than 50% of the body), forexample through entrance into the circulatory or lymph systems. Bycontrast, “local administration” refers to the introducing or deliveryto a subject an agent via a route which introduces or delivers the agentto the area or area immediately adjacent to the point of administrationand does not introduce the agent systemically in a therapeuticallysignificant amount. For example, locally administered agents are easilydetectable in the local vicinity of the point of administration but areundetectable or detectable at negligible amounts in distal parts of thesubject's body. Administration includes self-administration and theadministration by another. In one aspect, disclosed herein areanti-cancer combination therapies methods treating, preventing,reducing, and/or inhibiting a cancer of any preceding aspect; whereinthe at least one immunoregulatory molecule inhibitor is administeredsystemically and/or the oncodriver pulsed dendritic cell is administeredintratumorally.

It is understood and herein contemplated that while a singleadministration of the components of the disclosed anti-cancercombination therapies (i.e., the pulsed dendritic cells and/or theimmunoregulator molecule inhibitor) would be ideal, not every patientwill respond in the same manner Thus, in one aspect, disclosed hereinare anti-cancer combination therapies methods treating, preventing,reducing, and/or inhibiting a cancer; wherein the at least one pulseddendritic cell is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per dayor at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times perweek for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 weeks. Also disclosed herein areanti-cancer combination therapies methods treating, preventing,reducing, and/or inhibiting a cancer of any preceding aspect; whereinthe at least one immunoregulatory molecule inhibitor is administered atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, or 24 times per day or at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, or 14 times per week for at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks.It is further understood and herein contemplated that the order andduration of the administered components can vary as appropriate for thesubject being treated. In one aspect, disclosed herein are anti-cancercombination therapies methods treating, preventing, reducing, and/orinhibiting a cancer; wherein the pulsed dendritic cells are administeredat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3,4, 5, or 6 months prior to administration of the at least oneimmunoregulatory molecule inhibitor; are administered concurrently withthe at least one immunoregulatory molecule inhibitor; or wherein the atleast one immunoregulatory molecule inhibitor is administered at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, or6 months prior to administration of the pulsed dendritic cells.

As noted above, it is intended herein that the disclosed methods oftreating, inhibiting, reducing, and/or preventing cancer can augmentedwith any therapeutic treatment of a cancer including, but not limitedsurgical, radiological, and/or pharmaceutical treatments of a cancer. Asused herein, “surgical treatment” refers to tumor resection of the tumorby any means known in the art. Similarly, “pharmaceutical treatment”refers to the administration of any anti-cancer agent known in the artincluding, but not limited to Abemaciclib, Abiraterone Acetate,Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilizedNanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris(Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin(Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus),Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod),Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta(Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran forInjection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi(Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin(Chlorambucil), Amboclorin Chlorambucil), Amifostine, AminolevulinicAcid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex(Anastrozole), Aromasin (Exemestane),Arranon (Nelarabine), ArsenicTrioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi,Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine,Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq(Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa(Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, Bexxar (Tositumomaband Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine),Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif(Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel,Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx(Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, Campath(Alemtuzumab), Camptosar, (Irinotecan Hydrochloride), Capecitabine,CAPDX, Carac (Fluorouracil—Topical), Carboplatin, CARBOPLATIN-TAXOL,Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant,Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (DaunorubicinHydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab,CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine,Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar(Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate),Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen(Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP,Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine,Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide),Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin,Daratumumab, Darzalex (Daratumumab), Dasatinib, DaunorubicinHydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome,Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium),Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (CytarabineLiposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab,Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), DoxorubicinHydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (DoxorubicinHydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex(Fluorouracil—Topical), Elitek (Rasburicase), Ellence (EpirubicinHydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine,Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate,Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab),Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride,Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine),Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet(Doxorubicin Hydrochloride Liposome), Everolimus, Evista, (RaloxifeneHydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU(Fluorouracil Injection), 5-FU (Fluorouracil—Topical), Fareston(Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC,Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate),Fludarabine Phosphate, Fluoroplex (Fluorouracil—Topical), FluorouracilInjection, Fluorouracil—Topical, Flutamide, Folex (Methotrexate), FolexPFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB,FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil(Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPVNonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, GemcitabineHydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN,Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif(Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (CarmustineImplant), Gliadel wafer (Carmustine Implant), Glucarpidase, GoserelinAcetate, Halaven (Eribulin Mesylate), Hemangeol (PropranololHydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine,Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV QuadrivalentVaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea(Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib),Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride),Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride,Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide,Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate,Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic(Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin,Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A(Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab andTositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride,Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone,Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate),JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine),Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda(Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel),Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate,Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima(Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran(Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan(Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (DoxorubicinHydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and TipiracilHydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (LeuprolideAcetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib),Marqibo (Vincristine Sulfate Liposome), Matulane (ProcarbazineHydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate,Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride,Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide),Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide,Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, MitomycinC, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil(Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin(Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg(Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (PaclitaxelAlbumin-stabilized Nanoparticle Formulation), Navelbine (VinorelbineTartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), NeratinibMaleate, Nerlynx (Neratinib Maleate), Netupitant and PalonosetronHydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar(Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide,Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab,Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo(Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, OmacetaxineMepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride,Onivyde (Irinotecan Hydrochloride Liposome), Ontak (DenileukinDiftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin,Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD,Palbociclib, Palifermin, Palonosetron Hydrochloride, PalonosetronHydrochloride and Netupitant, Pamidronate Disodium, Panitumumab,Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin),Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim,Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b),Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab,Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide,Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza(Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride,Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (EltrombopagOlamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol(Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride,Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP,Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, RecombinantHuman Papillomavirus (HPV) Nonavalent Vaccine, Recombinant HumanPapillomavirus (HPV) Quadrivalent Vaccine, Recombinant InterferonAlfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH,Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE,Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human),Rituximab, Rituximab and, Hyaluronidase Human, Rolapitant Hydrochloride,Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride),Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, RuxolitinibPhosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc),Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate),Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, SterileTalc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), SunitinibMalate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b),Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid(Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc,Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine),Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna(Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq,(Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus,Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa,Tisagenlecleucel, Tolak (Fluorouracil—Topical), Topotecan Hydrochloride,Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin,Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride),Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide),Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), UridineTriacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride),Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade(Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta(Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (LeuprolideAcetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS(Vincristine Sulfate), Vincristine Sulfate, Vincristine SulfateLiposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (UridineTriacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (PazopanibHydrochloride), Vyxeos (Daunorubicin Hydrochloride and CytarabineLiposome), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib),Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yondelis(Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula(Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin(Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride),Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (GoserelinAcetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (ZoledronicAcid), Zydelig (Idelalisib), Zykadia (Ceritinib), and/or Zytiga(Abiraterone Acetate). Also contemplated herein are chemotherapeuticsthat are PD1/PDL1 blockade inhibitors (such as, for example,lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559,Atezolizumab, Durvalumab, or Avelumab).

Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,including topical intranasal administration or administration byinhalant. As used herein, “topical intranasal administration” meansdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines

Therapeutic Uses

Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptoms of the disorder are affected. The dosage should notbe so large as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any counterindications. Dosage can vary, and can be administered inone or more dose administrations daily, for one or several days.Guidance can be found in the literature for appropriate dosages forgiven classes of pharmaceutical products. For example, guidance inselecting appropriate doses for antibodies can be found in theliterature on therapeutic uses of antibodies, e.g., Handbook ofMonoclonal Antibodies, Ferrone et al., eds., Noges Publications, ParkRidge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies inHuman Diagnosis and Therapy, Haber et al., eds., Raven Press, New York(1977) pp. 365-389. A typical daily dosage of the antibody used alonemight range from about 1 μg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1: Anti-oncodriver Th1 Responses in Breast Cancer

Most breast tumors express oncodrivers including HER2, EGFR (ER+ andTNBC), C-MET (TNBC) and HERS (ER+, HER2, and TNBC). These oncodrivers ordownstream pathways are often targeted by many therapeutics and patientswith metastatic breast cancer (MBC) are often treated with targetedagents such as anti-estrogens, CDK4/6 inhibitors, HER2 directedtherapies and AKT inhibitors. Most of these patients, however, becomeresistant to therapies or stop responding and progress. Thus, thesepatients are in need of additional therapies. Checkpoint therapies haveshown promising but limited effectiveness in MBC so identifyingeffective new immunotherapies that may be combined with targeted agentsto make them more effective in MBC would be highly desirable.Oncodrivers may be critical appropriate targets of the immune responseas indicated by the data below.

Evidence for Loss of Anti-Oncodriver Th1 Response During Tumorigenesis

Healthy adult women possess unusually high, pre-existing Th1 immunityagainst HER2 (FIG. 1A and 1C, see, e.g., Dana J, et al., Oncoimmunology.2015;4:e1022301; Dana J, et al., Breast Cancer Res. 2015;17:71; andFracol, M., et al., Ann Surg Oncol (2017) 24: 407.https://doi.org/10.1245/s10434-016-5584-6) in the peripheral blood.These Tbet^(pos) Th1 cells, however, are progressively lost duringtumorigenesis. This deficit is first detectable at the ductal carcinomain situ (DCIS) stage and becomes profoundly suppressed by stage Iinvasive breast cancer (IBC) stage (FIG. 1A, 1C, and 1D). There is nosuch loss of HER2 Th1 immune responses during HER2^(neg) tumorigenesis(FIG. 1A), neither are there losses of responsiveness against other(non-tumor) control antigens (data not shown), indicating thissuppression is antigen-selective and not a product of global anergy.Standard therapy of surgery, radiation, and chemotherapy withtrastuzumab do not routinely correct this anti-HER2 Th1 loss, butvaccination with HER2-pulsed type I dendritic cells (DC1) candramatically increase peripheral anti-HER2 CD4 Th1, indicating this isnot a fixed deficit, but instead one that can be corrected byappropriate immunization (FIG. 1B). It is unclear whether thediminishment in peripheral blood CD4 Th1 signals a true loss of cells orrepresents a shift from central circulation to peripheral sites.

The Anti-HER2 Th1 Response Correlates with Clinical Outcomes

HER2^(pos) IBC patients achieving a pathological complete response (pCR)to neoadjuvant chemotherapy (NAC) demonstrate improved survival whilethose with residual disease at the time of surgery demonstrate increasedrisk of recurrence. Although invasive HER2^(pos) IBC patients showdepressed anti-HER2 Th1 immunity as a group, some individuals areprofoundly suppressed while others retain moderate responsiveness. Thosepatients that regain or partially retain anti-HER2 Th1 demonstratehigher pCR rate to neoadjuvant chemo/trastuzumab therapy, while thosewith residual disease display the lowest anti-HER2 CD4 Th1 responses(FIG. 1D). Shown is the loss of repertoire but there is also significantdifference in overall and cumulative responses. The loss ofanti-oncodriver CD4 immune response represents a decrease in interferongamma (IFN-γ) production in the Tbet^(pos) Th1 population and not GATA3^(pos) Th2 population, indicating the Th1 immune response may becritical to mediating anti-oncodriver BC responses. Indeed, elevatedimmune gene expression in the tumor is associated with improvedoutcomes. We have also investigated the relationship between anti-HER2immunity and disease-free survival (DFS). Patients with the recurrenceand reduced DFS display the most diminished anti-HER2 Th1 immuneresponse (FIG. 1F) while those with retained peripheral anti-HER2 Th1response have substantially greater DFS (FIG. 1F). This deficit in theperipheral CD4Th1 may reflect immunologic activity away from theperiphery or true loss of tolerized or exhausted response.

Example 2: Boosting anti-HER-2 CD4 Th1 Using DC1 Therapy in HER2^(pos)Early Breast Cancer

We have now conducted four clinical trials: two in patients withHER2^(pos) DCIS and two in patients with HER2^(pos) IBC. See: LowenfeldL, et al. Clin Cancer Res. 2017;23:2961-71. All four studies havedocumented that we can increase the anti-HER2 CD4 Th1 response inperipheral blood with DC vaccine administration which does not reallycorrelate with response to therapy but does demonstrate thatadministration of DC1 boosts anti-HER2 CD4 Th1. The DCIS patientsreceived just four-six weekly vaccines either in distant groin lymphnodes or in the region of the DCIS in the breast, while the invasivepatients received the initial six weekly intranodal followed by threemonth intranodal boosts ×3. Eighty five percent of the patientsdemonstrated an increase in anti-HER2 CD4 Th1. In the two DCIS studieswhere DC1 were administered in the neoadjuvant setting, about 30% of theER^(neg) patients demonstrated a pCR to DC1 vaccinations (FIG. 2A) andfor the ERPOS HER2^(pos) patients, adding anti-estrogen therapy with DC1vaccination improved the pCR rate to similar level (FIG. 2A), fromminimal background response indicating the efficacy of the combinationapproach. DC1 therapy was effective independent of whether administeredin distant nodes or in the DCIS region in the breast. Interestinglythose who achieved pCR demonstrated the highest levels of anti-HER2 CD4Th1 in sentinel nodes (FIG. 2B). This indicates that achieving asignificant anti-HER2 CD4 Th1 response in the local regional area of thetumor has the strongest clinical response and prompted us to study thisfurther in preclinical models leading to our current effective therapyof DC1. Although there are low numbers of subjects achieving a pCR inDCIS from DC1 therapy it portends a very favorable reduction in anysubsequent breast events ((SBE), FIG. 2C) compared to those achieving<pCR, something that would be critical to reduce recurrence and reducemortality in high risk DCIS patients. We have studied the impact of DC1therapy and Th1 cytokines on disseminated cancer cells (DCC) as that maybe crucial to reduce mortality. For patients with IBC T1a/b lesions intheir DCIS there was only about 8% incidence of finding no residualdisease at surgery. It is clear that this therapy has shown promise butneeded to be improved to be developed as satisfactory therapy for thegroup of patients with HER2 expressing DCIS and T1a/b IBC. Thus, wereturned to preclinical models to build upon this opportunity.

Example 3: DC1 Vaccines in Combination with SEMA4D Blockade

Semaphorin 4D (SEMA4D) is a member of family of cell surface moleculesthat are essential for tissue and organ development and are involved inimmune regulation. Antibodies against SEMA4D have been shown to regulatelymphocyte infiltration into tumors (see, e.g., U.S. Pat. No.9,243,068). In this Example, we combined an anti-SEMA4D monoclonalantibody (Mab 67, see, e.g., U.S. Pat. No. 8,496,938, with DC1vaccinations in a HER2 murine TUBO tumor model as follows. The TUBOmouse mammary tumor cell line (Accession No. CVCL_2A33, Rovero, S., etal., J. Immunol 165:5133-5142 (2000)) was first shown to express SEMA4Dby immunohistochemistry (FIG. 3A). For single tumor model (FIG. 3B1),Balb/C mice received 2.5e5 TUBO cells subcutaneously on right flank onday 0. When tumors were palpable on day 7, mice were randomized intofour groups. Mice received monotherapy with either control antibody oranti-sema4D antibody intraperitoneally until end point or intratumoralHER2-DC1 weekly for six weeks. For combination therapy, mice receivedanti-sema4D antibody prior to receiving first intratumoral HER2-DC1injection once a week for six weeks. For bilateral model, Balb/c mice(N=6) were injected at both flanks subcutaneously with 2.5×10⁵ tumorcells/ site on day 0. DC were generated, matured to DC1 as describedpreviously (Cintolo J A, 2016, Melanoma Res. 2016 February; 26(1):1-11)and pulsed with neu peptides. BALB/c mice received DC vaccinesintratumorally once a week for six weeks. In those animals with twotumor locations, that vaccine was administered in only one of the twotumors. Mab 67 was given intraperitoneally at the concentration of 10mg/kg/ body weight at weekly interval. Control mice received isotypecontrol antibodies, DC treatment or Mab 67 alone. Tumors were measuredevery 2-3 days with a caliper until the endpoint. For comparison of invitro measurements, a one-way ANOVA (followed by Tukey post hoc test)was performed. For comparison of in vivo measurements, the same test wasperformed using tumor measurement taken at each time point. AMann-Whitney test was used to compare between two treatment groups. Allstatistical evaluations of data were performed using GraphPad Prismsoftware. Statistical significance was achieved at p<0.05.

Systemic administration of the anti-SEMA4D antibody with intratumoralHER2 pulsed DC1 to mice bearing established TUBO tumors resulted incomplete tumor regression of the vaccine-treated tumor compared totreatment with the anti-SEMA4D antibody or the DC1 alone (FIG. 3B1).This translated to increased survival (FIG. 3B2). An intratumoralinjection of HER2 DC1 to a single tumor resulted in regression ofnon-injected contralateral tumors (FIG. 3C1 and 3C2). Further flowcytometric analyses demonstrated enrichment of anti-HER2 CD4 Th1responses including reduced myeloid-derived suppressor cell tumorinfiltration (FIG. 3E), increased CD4 T cell tumor infiltration (FIG.3F), and increased CD4 T cell lymph node infiltration (FIG. 3G). Thepotential relevance to human tumors is highlighted by the fact thatSEMA4D was expressed in about 60% of human HER2 DCIS or HER2 IBC asmeasured by immunohistochemistry (FIG. 3D).

Example 4: Administration of Intratumoral HER2 Pulsed DC1 and SystemicAnti-SEMA4D Antibody Resulted in Strong Anti-Tumor Th1 Responses

Spleens were harvested from mice that were rendered cured of TUBO tumorsby the combination systemic administration of anti-SEMA4D antibody withintratumoral HER2 pulsed DC1 therapy as described in Example 3, as wellfrom control mice vaccinated intratumorally with the DC1 vaccine aloneor in combination with anti-SEMA4D antibody, and CD4+ T cells wereisolated from splenocytes were co-cultured with with HER2 pulsed DC1 forthree-four days followed by expansion in the presence of IL-2 (10 U/ml)and IL-7 (20 ng/ml for three weeks. Cells expanded from spleensharvested from mice vaccinated with HER2 pulsed DC1 either alone or incombination with systemic anti-SEMA4D antibody treatment were compared(FIG. 4). The data indicate the CD4 T cells from mice given combinationtherapy displayed an in vitro expansion of about 7-fold compared to thatof the control mice of about 3-fold (FIG. 4A). The CD4 T cells expandedfrom the spleens of mice given the combination therapy also displayedstrong antigen specificity for HER2/neu-derived peptides p5, p435, andp1209 (see, e.g., Jalali et al., Nanomedicine; 8:692-701 (2012)) ascompared to the CD4 T cells expanded from mice that received the DC1vaccine alone (FIG. 4B). Moreover, the CD4 T cells expanded from thespleens of mice given the combination therapy displayed a strongproduction of IFN-γ indicating a strong TH1 response (FIG. 4C). Theseresults show that CD4 T cells from HER2-DC1+aSEMA4D treated mice weresuperior in proliferation, function and specificity compared to T cellsfrom HER2-DC1 alone vaccinated mice.

Example 5: DC1 Treatment of BALB/c Neu T Transgenic Mice ImpactsDisseminated Cancer Cells in HER2 Expressing Mammary Carcinoma

The DC1 vaccines were tested in HER2 patients with residual diseasefollowing neoadjuvant chemotherapy/trastuzumab, the DC1 administrationwas safe and resulted in increased anti-HER2 CD4 Th1 and there have beenno recurrences in 17 patients with median follow-up of 40 months. Sincemany of these patients destined to recur would be harboring disseminatedcancer cells (DCC), we wanted to study the effects of Th1 cytokines andas well as CD4 Th1 cells on DCC. For this we utilized the BALB/c Neu Ttransgenic mouse model. NeuT is a transgenic mouse model of breastcancer in which the mouse mammary tumor virus (MMTV) promoter drivesneuT expression and mice develop spontaneous tumors in the mammary fatpads of female mice. When female BALB-neuT mice reach 21-28 d of age,the neuT protein is overexpressed in mammary glands and areas ofatypical hyperplasia start to form, which progress to in situ carcinomasat about day 60 and to invasive cancers by day 120-150 Neoplastic changeoccurs, albeit asynchronously, in all mammary glands so that by aboutday 120, one or more tumors are palpable and by about day 230 all 10mammary glands contain palpable tumors. We and others have demonstratedthere are disseminated cancer cells that can be identified in severalorgans prior to the appearance of mammary carcinomas (FIG. 5A and 5B).These HER2+ cytokeratin positive cells can be detected in the bonemarrow, lungs and liver from week 9 on and they compose 10-15% of thecells and are in a reasonably low proliferative state as measured byKi67 (FIG. 5C). Vaccination of mice with HER2 pulsed DC1 after week 9when DCC are present but prior to the development of mammary carcinomaresulted in a decrease in DCC as well as a decreased expression of Ki67(FIG. 6A). DC1 Vaccines reduced the development of mammary carcinoma atweek 16 (FIG. 6B). There was also a diminished number of DCC (FIG. 6C)and increase in markers of tumor senescence in DCC from Neu T micetreated with DC1. Shown in FIG. 6D is β-galactosidase expression acommon marker of senescence. This latter data indicates that, besidesthe impact of therapy on primary or metastatic disease, withintratumoral therapy, DC1 can through induction of anti-HER2 CD4 T cellsdrive senescence and protect against tumor development and may have animpact on BC mortality.

Example 6: Immune Cell Infiltration After DC1 or SEMA4D Treatment

BALB-neu transgenic (Neu T) mice were treated at 8 weeks of age withsubcutaneous injections of 1×10⁶ rat neu peptide-pulsed DC1 twice a weekor with anti-SEMA4D antibody administered weekly at a concentration of10 mg/kg/body weight. On week 16, the mice were sacrificed, and mammaryglands were collected. The single cell suspension of mammary glands wasprepared and stained for CD19 positive B cells then analyzed by flowcytometry. An increased level of CD19+ B cells was observed in DC1vaccinated mice and in anti-SEMA4D antibody-treated mice compared tountreated control, indicating B cell infiltration into the mammaryglands (FIG. 7A). The accumulation of B cells in the tumor regionindicates that antitumor antibodies play a role in causing the effectsseen in DC1 therapy with SEMA4D. In addition, the bone marrow cellsderived from the control and DC1 vaccinated NeuT mice were stained forCD4 and CD8 T cell markers and analyzed by flow cytometry. Increasednumbers of CD4T cells and CD8 T cells were observed in the bone marrowof DC1 vaccinated NeuT mice compared to untreated control mice (FIG.7B).

Example 7: IFN-γ and TNF-α from CD4 Th1 Cause Induction of TumorSenescence in HER2 Expressing Breast Cancer

Since CD4 and B cells (MHC class II APC) accumulate around the DCISducts, we investigated the impact of Th1 cytokines on HER2 BC cells.IFN-γ and TNF-α cause the induction of both apoptosis and tumorsenescence in HER2 expressing BC cells. The therapeutic benefit ofblocking HER2/HERS signaling in breast cancer has been demonstrated inboth in vitro studies and clinically. We explored the senescent andapoptotic effects of Th1 cytokines in high and intermediateHER2-expressing cell lines blocked with HER2 and HER3 siRNA (FIG. 2).Although the combined treatment of TNF-α and IFN-γ in HER3-knocked downSK-BR-3 cells did not significantly enhance the number of senescentcells, higher SA-β-gal staining was observed in cells treated with dualHER2/HER3-knocked down combined with Th1 cytokines (FIG. 8A).Trastuzumab, a humanized recombinant monoclonal antibody directedagainst the extracellular subdomain IV of HER2, inhibitsligand-independent dimerization, blocks downstream proliferationsignaling pathways, and induces antibody-dependent cellular cytotoxicity(ADCC) and Pertuzumab, another humanized recombinant monoclonal antibodytargeting the extracellular subdomain II of HER2, preventsligand-dependent heterodimerization with other members of the HERfamily, which also inhibits proliferation signaling pathways and inducesADCC. Together both antibodies act in a complementary fashion. Weapplied our paradigm of Th1 cytokine-induced senescence and apoptosis toa combination model, using TNF-α and IFN-γ treatment together withtrastuzumab and pertuzumab. In HER2high SK-BR-3 cells, we found thatsenescence increased synergistically in cells treated with thecombination of cytokine and antibodies compared to untreated cells,cells treated with cytokines alone, or cells treated with antibodiesalone, as measured by SA-β-gal staining (FIG. 8B, p<0.001) andp15^(INK4b) expression(Cyclin-dependent kinase 4 inhibitor B, also knownas p15^(INK4b)) (FIG. 8C). Notably, the combined treatment not onlyinduced a relatively higher percentage of blue senescent cells, butthere were also significantly fewer cells overall. Increased apoptosisin an additive fashion was demonstrated by increased active caspase-3expression (FIG. 8C) and increased annexin V and propidium iodidepositive cells (FIG. 8D & E). We explored the potential for TNF-α andIFN-γ to induce senescence and apoptosis in trastuzumab and pertuzumabresistant cell lines. HCC-1419 and JIMT-1 cells were treated withtrastuzumab and pertuzumab did not induce senescence or apoptosis (FIG.9). However, the dual treatment with cytokines and targeted therapiesinduced significantly greater senescence as evidenced by SA-β-gal assay(FIG. 9A) and increased expression of p15^(INK4b) in HCC-1419 cells(FIG. 9B) and JIMT-1 cells (FIG. 9C). Th1 cytokines combined withHER2/HER3 blockade can cause tumor senescence and apoptosis even in celllines resistant to trastuzumab and pertuzumab.

We next tested whether anti-HER2 CD4 Th1 cells could impact HER2^(pos)BC cells separated by transwell membrane. SK-BR-3 breast cancer cellsco-cultured with CD4+ T-cells from breast cancer patients primed withClass II HER2 peptides resulted in senescence and apoptosis of SK-BR-3cells, evidenced by increased SA-β-gal staining (FIG. 10A) andp15^(INK4b) and cleaved caspase-3 expression (FIG. 10B, CD4+− DC H, 3).CD4+ T-cells primed either with immature dendritic cells (CD4+− IDC H(2)) or mature DCs plus irrelevant Class II peptides (BRAF: CD4+− DC B(5); or survivin: CD4+− DC S (6)) were not able to induce senescence orapoptosis of SK-BR-3 cells. Similar to the previously demonstratedsynergistic effect, senescence and apoptosis were significantlyaugmented when trastuzumab and pertuzumab (4) were added to the culture,evidenced by increased SA-β-gal staining (FIG. 10A, p<0.001) andp15^(INK4b) and cleaved caspase-3 expression (FIG. 10B, CD4+− DC H TP,4). These results indicate that the anti-HER2 CD4 Th1 cells do not needto interact directly with the tumor cells but rather antigen presentingcells (APC) in the TME, and the Th1 cytokines can synergize with HER2targeted therapy. This may form the basis of having DC1 pulsed and boundin the tumor environment to drive anti-HER2 CD4 Th1 responses and thepossibility that antibodies like HP can synergize with Th1 cytokines toeliminate tumor cells.

Example 8: Clinical Activity of Th1 Cytokines (IFN-γ) in HER2 BreastCancer

We have built on preclinical findings by developing a Phase I/II studyinvestigating whether IFN-γ can be administered safely and have effectsin clinical response. A Phase I study demonstrated IFN-γ administeredsubcutaneously three times a week with THP in patients with first linemetastatic BC was safe and resulted in disease stabilization of partialresponses (FIG. 11). This has now progressed to a Phase II study beingadministered in a neoadjuvant setting to those with >T2 ER+HER2+ IBC. 15patients are now enrolled and two of the first four patients have had apCR. We are encouraged by this as this subtype of patients treated withPTCH, a significantly more toxic regimen, demonstrate about 27% pCRrate. As shown herein, this regimen is successful in at leastmaintaining the established pCR rate and thus, the regimen can replacethe more toxic regimens with simpler potentially more effective regimenusing a combination of immunotherapy with Th1 cytokines and standardtherapy. We are determining whether DC1 vaccines can drive a similarresponse in neoadjuvant HER2 patients to drive pCR in combination withPTCH. These data point to the clinical benefits of Th1-cytokines likeIFN-γ to impact HER2 tumors and are poised to become components of thetrue non-chemotherapy regimens in HER2 BC.

Example 9: Accumulation of B and T Cells Occurs in Patients Respondingto HER2-pulsed DC1 Vaccines

Although patients with pCR demonstrate no residual DCIS ducts in thebreast, many of those with residual disease affords the opportunity toassess the response in the breast of those with residual disease. Theresponse in the breast is often heterogeneous with areas of denselymphocytic infiltrate (FIG. 12A & B) and areas with little or noresponse (FIG. 12C & D). The accumulation of lymphocytes represents alarge fraction of CD4 T cells as well as CD20+ B cells and some CD8 Tcells (FIG. 13). These structures mimic ectopic lymphoid structureswhich are associated with favorable responses. In patients withsignificant accumulation of these lymphoid structures in and around DCISducts we often see dying or dead ducts, indicating the immune responseis causing death and elimination of DCIS (FIG. 14).

Effect of Anti-SEMA4D Antibody on the TME and Tumor Vasculature

SEMA4D has been described as a promoter of angiogenesis of tumors and,therefore, has been associated with tumor progression (see, e.g., Zhou,et al. Methods Mol. Biol 1493:429-441 (2017)). To study how SEMA4Dmodifies TME, specifically tumor vascularity, we observed tumorvascularity in mice injected with TUBO cells as described in Example 3by in-vivo MRI. Dynamic Contrast Enhancement (DCE)—MRI were used toexamine different properties of tumor vascularity: Using Area undercurve (AUC), slope, time to maximum, we determined the status of tumorvascularity. We injected 0.2 mmol/kg of Gadavist through the tail veinto investigate how single treatments with DC1 alone or anti-SEMA4D aloneor both agents in combination affect the tumor vascularity andregression of tumor. We observed smaller slope, less accumulation of thecontrast agent, and less vessel leakage (FIG. 15A&B) in mice thatreceived combination therapy compared to untreated or monotherapy. Thisdata indicates that combination therapy with intratumoral DC1 withsystemic administration of an anti-SEMA4D antibody improves tumorvascularity indicated by smaller slope and less accumulation of thecontrast agent and less vessel leakage which may play a role in theinducing complete tumor regression in both single and bilateral TUBOmodel.

Example 10: Effect of Anti-SEMA4D on Tumor pH

Effect of anti-SEMA4D on tumor pH:. Tumors have been shown to be acidicand tumor acidosis has been correlated with poor prognosis and greatlyrelies on vascularity to reduce their acidity, we examined tumor pH bymeans of Chemical Exchange Saturation Transfer (CERST)-MRI experiments.As shown in FIG. 16, we observed that treatment with SEMA4D alone had aneffect on shifting tumor pH from acidic to alkaline over time.

The murine monoclonal antibody Mab 67, described above, is disclosed,e.g., in U.S. Pat. No. 8,496,938.

The amino acid sequences of the MAb 67 VH and VL genes are shown belowwith the CDR1, CDR2 and CDR3 regions underlined.

MAb 67 VH: (SEQ ID NO: 1)QVQLQQSGPELVKPGASVKISCKASGYSFSDYYMHWVKQSPENSLEWIGQINPTTGGASYNQKFKGKATLTVDKSSSTAYMQLKSLTSEESAVYYCTRYY YGRHFDVWGQGTTVTVSSMAb 67 VL: (SEQ ID NO: 2)DIVMTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPY TFGGGTKLEIK

1. Mab VX15/2503 (pepinemab) is a humanized version of MAb 67, and isalso disclosed in U.S. Pat. No. 84,969,38. The amino acid sequences ofVX15/2503 are reproduced below.

Sequence of VX15/2503 VH: (SEQ ID NO: 3)QVQLVQSGAEVKKPGSSVKVSCKASGYSFSDYYMHWVRQAPGQGLEWMGQINPTTGGASYNQKFKGKATITVDKSTSTAYMELSSLRSEDTAVYYCARYY YGRHFDVWGQGTTVTVSSSequence of VX15/2503 VL: (SEQ ID NO: 4)DIVMTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPY TFGQGTKLEIK

Example 11: Depletion of CD4+ T Cells Abrogates Anti-SEMA4D Activity,Both Alone and in Combination with DC-1 Treatment

As a whole, CD4+ T cells play a major role in instigating and shapingadaptive immune responses. To study whether CD4+T cells are necessaryfor a clinical response to combination HER2 DC1 vaccine/anti-SEMA4Dtreatment, CD4 depleted tumor-bearing mice were generated and the immuneresponse to treatment was compared to that of non-CD4 depleted(“normal”) tumor bearing mice.

Balb/C mice received 2.5e5 TUBO cells subcutaneously on the right flankon day 0. When tumors were palpable on day 7, the mice were randomizedinto four groups. Mice received monotherapy with either an IgG isotypecontrol antibody or anti-SEMA4D antibody/MAb67 (10 mg/kg/body weight)intraperitoneally until end point or 1e6/100 μl intratumoral HER2-DC1weekly for six weeks. For combination therapy, mice received anti-sema4Dantibody prior to receiving a first intratumoral HER2-DC1 injection oncea week for six weeks.

CD4 depleted Balb/C mice were generated by administering 300 μg ofanti-CD4 depleting antibody (Clone GK1.4) intraperitoneally, startingthree days before TUBO tumor implantation, and treatment with theanti-CD4 antibody continued with two injections per week until endpoint. (See Evans, E. E., et al. (2015). Cancer Immunol Res 3(6):689-70) The CD4-depleted mice were treated with monotherapy, anti-SEMA4Dantibody/MAb67, or combination therapy as described above fortumor-bearing non-CD4 depleted mice.

Mean tumor volume and survival for each group are shown in FIG.17A-D-17A and B (Control, no CD4 depletion; tumor volume and survival,respectively), and 17C and D (CD4 depletion). FIGS. 17E (control, no CD4depletion) and 17F (CD4 depletion) show tumor growth curves for eachmouse. Complete tumor regression was observed following treatment withanti-SEMA4D and activity was further enhanced with the combinationtherapy. All of the combination-treated mice in the non-depleted groupsurvived for the sixty-day testing period (FIG. 17B), and 4/5 of thesemice showed complete tumor regression. (FIG. 17E). However, depletion ofCD4+ T cells completely abrogated activity of anti-SEMA4D treatment,both alone and in combination with the DC-1 vaccine (FIG. 17D). Thesedata indicate that CD4+ T cells are required for a clinically effectiveresponse to anti-SEMA4D therapies.

Example 12. Role of Fc Gamma Receptors (FcγR) on Clinical Effect ofCombination Therapy

The FcγR mediated functions most commonly associated with therapeuticantibodies are those that mediate target cellelimination—antibody-dependent cellular cytotoxicity (ADCC), which is amechanism of cell-mediated immune defense whereby an effector cell ofthe immune system actively lyses a target cell, whose membrane-surfaceantigens have been bound by specific antibodies. These functions aretriggered when antibody binding to antigen on the surface of a targetcell generates sufficient avidity to trigger signaling through FcγRs oneffector cells such as NK cells and macrophages, which then eliminatetarget cells through direct killing or phagocytosis. Preclinical modelsshow that these forms of FcγR-mediated cytotoxicity are a significantcomponent of the mechanism of action for certain tumor targetedantibodies. (Clynes R A et al., Inhibitory Pc receptors modulate in vivocytoxicity against tumor targets. Nat Med. 2000, 6: 443-446).

To study the role of FcγR in the clinical response to the combinationtherapy, FcγR knock-out mice were generated. Briefly, the model wascreated by targeted disruption of the Fcer1g gene via introduction of anew stop codon in E14 ES cells and injecting the targeted cells intoC57BL/6 blastocysts. Heterozygotes on a C57BL/6 background wereintercrossed to generate homozygous targeted mutation mice. The micewere then backcrossed twelve generations (N12) to a BALB/cByJ inbredbackground. (Takai T, Li M, Sylvestre D, Clynes R, Ravetch J. (1994)FcRγ Chain Deletion results in Pleiotrophic Effector Cell Defects.).

BALB/C-Fcer1g KO mice (C.129P2(B6)-Fcer1g^(tm1Rav) N12) were injected atboth flanks subcutaneously with 2.5×10⁵ tumor cells/ site on day 0. DCwere generated, matured to DC1 and pulsed with MHC class II neupeptides. Mice received 1e6/100 μ1 DC1 vaccine intratumorally once aweek for six weeks. In those animals with two tumor locations, thevaccine was administered in only one of the two tumors. Anti-SEMA4D Mab67 was given intraperitoneally at a concentration of 10 mg/kg/bodyweight at weekly intervals. Control mice received isotype controlantibodies, DC treatment or Mab 67 alone. Tumors were measured every 2-3days with a caliper until the endpoint. Mean tumor volume of treatedtumors and survival for each group are shown in FIG. 18A and 18B,respectively. Tumor growth curves for each mouse are shown in FIG. 18C.(CR=complete tumor regression; tumor volume <50 mm²).

The data show that in mice lacking Fc Receptor gamma expression, thecombination therapy significantly delayed tumor growth compared to notreatment or single agent treatment (FIG. 18A and B). However, completetumor regression was not achieved (FIG. 18B), in contrast to controlmice treated with combination therapy (FIG. 17E), which indicates thatFc Receptor gamma is essential for complete anti-tumor efficacy of thecombination therapy.

Example 12. Role of IFN-γ on the Clinical Effect of Combination Therapy

Interferon-gamma (IFN-γ) is a pleiotropic molecule with associatedantiproliferative, pro-apoptotic and antitumor mechanisms. This effectorcytokine is considered to be a major effector of immunity and is a keyTh1 cytokine relevant for anti-tumor immune response.

To study the role of IFN-γ in the clinical response to the combinationtherapy, tumors were generated in Balb/C IFN-gamma knock out (KO)(C.129S7 (B6)-IFNg^(Tm1Ts)/J (IFN-γ^(KO), Jackson Laboratories) mice.The mice were administered 2.5e5 TUBO cells subcutaneously on the rightflank on day 0. Dendritic cells were generated, matured to DC1 andpulsed with MHC class II neu peptides. On day 7 when tumors werepalpable, mice were randomized into four groups. Mice receivedmonotherapy with either control antibody or anti-sema4D antibody (10mg/kg/body weight) intraperitoneally until end point or 1e6/100 μlintratumoral HER2-DC1 weekly for six weeks. For combination therapy,mice received anti-sema4D antibody prior to receiving a firstintratumoral HER2-DC1 injection once a week for six weeks. Tumors weremeasured every 2-3 days with a caliper until the endpoint. Mean tumorvolume for each group is shown in FIG. 19.

The data demonstrate that anti-tumor activity of DC1, anti-SEMA and thecombination requires IFN-γ for activity. These results are similar tothose obtained with CD4 depletion of immunocompetent mice (FIG. 17),demonstrating that both IFN-γ and CD4 cells are necessary for anti-tumorresponse for ami-SEMA4D therapies.

What is claimed is:
 1. An anti-cancer combination therapy comprising atleast one dendritic cell pulsed with an oncodriver and at least oneimmunoregulatory molecule inhibitor; wherein the immunoregulatorymolecule comprises Semaphorin 4D or VEGF.
 2. The anti-cancer combinationtherapy of claim 1, wherein the oncodriver is human epidermal growthfactor receptor (HER) HER2,
 3. The anti-cancer combination therapy ofclaim 1 or claim 2, wherein the oncodriver pulsed dendritic cell isactivated with IL-12 prior to administration.
 4. The anti-cancercombination therapy of any one of claims 1 to 3, wherein theimmunoregulatory molecule inhibitor comprises an antagonist antibodythat specifically binds to SEMA4D or VEGF.
 5. The anti-cancercombination therapy of claim 4, wherein the antagonist antibody thatspecifically binds to SEMA4D comprises pepinemab.
 6. A method oftreating a cancer in a subject comprising administering the anti-cancercombination therapy of any one of claims 1 to
 5. 7. The method oftreating a cancer of claim 6, wherein the at least one immunoregulatorymolecule inhibitor is administered systemically.
 8. The method oftreating a cancer of claim 6, wherein the oncodriver pulsed dendriticcell is administered intratumorally.
 9. A method of treating a cancer ina subject comprising administering to the subject an oncodriver pulseddendritic cell and at least one immunoregulatory molecule inhibitor,wherein the immunoregulatory molecule comprises Semaphorin 4D or VEGF.10. The method of claim 9, wherein the oncodriver is human epidermalgrowth factor receptor (HER), HER2.
 11. The method of claim 9 or claim10, wherein the immunoregulatory molecule inhibitor comprises anantagonist antibody that specifically binds to SEMA4D or VEGF.
 12. Themethod of claim 11, wherein the antagonist antibody that specificallybinds to SEMA4D comprises pepinemab.
 13. The method of any one of claims9 to 12, wherein the dendritic cells are removed from the subject andpulsed with oncodriver ex vivo.
 14. The method of any one of claims 9 to13, wherein the pulsed dendritic cells are administered intratumorally.15. The method of any one of claims 9 to 14, wherein the immunoregulatormolecule inhibitor is administered systemically.
 16. The method of anyone of claims 9 to 15, wherein the pulsed dendritic cells areadministered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30,36 hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45days, 2, 3, 4, 5, or 6 months prior to administration of the at leastone immunoregulatory molecule inhibitor.
 17. The method of any one ofclaims 9 to 15, wherein the pulsed dendritic cells are administeredconcurrently with the at least one immunoregulatory molecule inhibitor.18. The method of any one of claims 9 to 15, wherein the at least oneimmunoregulatory molecule inhibitor is administered at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, or 6 monthsprior to administration of the pulsed dendritic cells.
 19. The method ofany one of the preceding claims wherein the cancer is breast cancer,melanoma, colorectal cancer, pancreatic cancer, prostate cancer, bladdercancer, ovarian cancer, stomach cancer, any combination thereof, or anymetastasis thereof.